ABSTRACT
Immunogenicity of 12 capsular polysaccharides (CPS) from Streptococcus pneumoniae did not correlate with pre-existing levels of natural IgM anti-CPS antibodies in mice. Immunization of mice with individual CPS, with the exception of type 14 (the only neutral CPS tested), increased serum IgM that also bound other CPS serotypes independent of structural similarity or commonly known contaminants. Surprisingly only IgM response to type 4 (which has a small immunodominant epitope) was dependent on either complement C3 or complement receptors CD35/CD21. IgG anti-CPS responses were infrequently induced, but critically dependent on complement. Our results have clarified the role of complement in the induction of IgM and IgG anti-CPS antibody responses in mice and have implications for CPS vaccine development.
Subject(s)
Antibodies, Bacterial/immunology , Antibody Specificity , Bacterial Capsules/immunology , Complement System Proteins/immunology , Immunoglobulin M/immunology , Animals , Antibodies, Bacterial/blood , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Female , Immunodominant Epitopes/immunology , Immunoglobulin M/blood , Male , Mice , Mice, Inbred C57BL , Streptococcus pneumoniae/immunologyABSTRACT
We examined the carbohydrate-binding potential of the C-type lectin-like receptor Dectin-2 (Clecf4n). The carbohydrate-recognition domain (CRD) of Dectin-2 exhibited cation-dependent mannose/fucose-like lectin activity, with an IC(50) for mannose of approximately 20 mM compared to an IC(50) of 1.5 mM for the macrophage mannose receptor when assayed by similar methodology. The extracellular domain of Dectin-2 exhibited binding to live Candida albicans and the Saccharomyces-derived particle zymosan. This binding was completely abrogated by cation chelation and was competed by yeast mannans. We compared the lectin activity of Dectin-2 with that of two other C-type lectin receptors (mannose receptor and SIGNR1) known to bind fungal mannans. Both mannose receptor and SIGNR1 were able to bind bacterial capsular polysaccharides derived from Streptococcus pneumoniae, but interestingly they exhibited distinct binding profiles. The Dectin-2 CRD exhibited only weak interactions to some of these capsular polysaccharides, indicative of different structural or affinity requirements for binding, when compared with the other two lectins. Glycan array analysis of the carbohydrate recognition by Dectin-2 indicated specific recognition of high-mannose structures (Man(9)GlcNAc(2)). The differences in the specificity of these three mannose-specific lectins indicate that mannose recognition is mediated by distinct receptors, with unique specificity, that are expressed by discrete subpopulations of cells, and this further highlights the complex nature of carbohydrate recognition by immune cells.
Subject(s)
Lectins, C-Type/chemistry , Lectins, C-Type/metabolism , Mannose-Binding Lectins/metabolism , Mannose/chemistry , Mannose/metabolism , Binding, Competitive , Candida albicans/metabolism , Humans , Inhibitory Concentration 50 , Lectins, C-Type/genetics , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Solubility , Zymosan/metabolismABSTRACT
The cysteine-rich domain (CR) of the mannose receptor binds sulfated glycoprotein CR ligand (CRL) expressed by subpopulations of myeloid cells in secondary lymphoid organs (CRL(+) cells). In naïve mice, these CRL(+) cells, metallophilic macrophages (M) in spleen and subcapsular sinus M in lymph nodes, are located strategically for antigen capture and are adjacent to B cell follicles, but their role in the immune response is unknown. We have exploited the lectin activity of CR to develop a highly specific system for targeting protein to CRL(+) M. We demonstrate that the sulfated carbohydrates recognized by CR are exposed to the extracellular milieu and mediate highly specific targeting of CR-containing proteins. This model will allow the dissection of the role of metallophilic M in an immune response in vivo.
Subject(s)
Macrophages/immunology , Macrophages/metabolism , Metals/metabolism , Proteins/metabolism , Animals , Cattle , Complement Activation , Complement System Proteins/immunology , Cysteine/analysis , Cysteine/metabolism , Humans , Immunoglobulin Fc Fragments/immunology , Lectins/metabolism , Lectins, C-Type/chemistry , Lectins, C-Type/genetics , Lectins, C-Type/metabolism , Ligands , Luteinizing Hormone/metabolism , Mannose Receptor , Mannose-Binding Lectins/chemistry , Mannose-Binding Lectins/genetics , Mannose-Binding Lectins/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Mutant Strains , Mutation/genetics , Protein Binding , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Substrate SpecificityABSTRACT
The study of the murine macrophage mannose receptor (MR) has been hampered by the lack of specific reagents. We have generated and characterized novel anti-MR monoclonal antibodies and used them to analyze MR expression in primary mouse macrophages (M(phi)). In BioGel- and thioglycollate-elicited M(phi), interleukin (IL)-4 up-regulated total cell-associated MR (cMR), correlating with enhanced surface expression. We investigated the influence of IL-10, a well-characterized deactivator of M(phi) function, on MR levels and observed that it had a similar effect to IL-4. In both cases, enhanced cMR levels translated into increased production of the soluble form of the receptor (sMR). We have demonstrated the presence of sMR in cultures of stable non-M(phi) transductants expressing full-length MR, indicating that the proteolytic activity responsible for cMR cleavage is not M(phi)-restricted. These data support a role for the MR in T helper cell type 2 cytokine-driven, immune responses and suggest a non-M(phi) contribution to sMR production in vivo.
Subject(s)
Antibodies, Monoclonal/pharmacology , Interleukin-10/pharmacology , Interleukin-4/pharmacology , Lectins, C-Type/biosynthesis , Macrophages, Peritoneal/drug effects , Mannose-Binding Lectins , Receptors, Cell Surface/biosynthesis , 3T3 Cells , Animals , Antibodies, Monoclonal/immunology , Blotting, Western , CHO Cells , Cell Line , Cricetinae , Cricetulus , Endocytosis , Flow Cytometry , Lectins, C-Type/genetics , Lectins, C-Type/immunology , Macrophages, Peritoneal/metabolism , Mannose/metabolism , Mannose Receptor , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Receptors, Cell Surface/genetics , Receptors, Cell Surface/immunology , Recombinant Fusion Proteins , Serum Albumin/metabolism , Solubility , Specific Pathogen-Free Organisms , Th2 Cells/immunology , Transduction, Genetic , Up-Regulation/drug effectsABSTRACT
The in vitro binding of the macrophage mannose receptor to a range of different bacterial polysaccharides was investigated. The receptor was shown to bind to purified capsular polysaccharides from Streptococcus pneumoniae and to the lipopolysaccharides, but not capsular polysaccharides, from Klebsiella pneumoniae. Binding was Ca(2+)-dependent and inhibitable with d-mannose. A fusion protein of the mannose receptor containing carbohydrate recognition domains 4-7 and a full-length soluble form of the mannose receptor containing all domains external to the transmembrane region both displayed very similar binding specificities toward bacterial polysaccharides, suggesting that domains 4-7 are sufficient for recognition of these structures. Surprisingly, no direct correlation could be made between polysaccharide structure and binding to the mannose receptor, suggesting that polysaccharide conformation may play an important role in recognition. The full-length soluble form of the mannose receptor was able to bind simultaneously both polysaccharide via the carbohydrate recognition domains and sulfated oligosaccharide via the cysteine-rich domain. The possible involvement of the mannose receptor, either cell surface or soluble, in the innate and adaptive immune responses to bacterial polysaccharides is discussed.
